This laboratory is appropriately titled Translational Research, as we use inherited retinal degenerations identified in the clinic as both a source of clues about retinal function and dysfunction and a target for research in therapeutic intervention. We have studied a number of mouse and rat models of human retinal degeneration diseases to elucidate the mechanisms of retinal neural signaling deficiencies and degeneration leading to blindness. We have investigated the role played by a number of retinal in proteins in the disease phenotypes and their possible use in treatment. We use normal rodents and rodents that are genetically altered to mimic human retinal disease to study the characteristics (phenotype), molecular genetics, physiological mechanisms and possible treatments of inherited retinal degenerations. Our laboratory applies the techniques of light and electron microscopy, immunohistochemistry, biochemistry, and molecular biology to human and animal retinal tissue, as well as the electroretinogram (ERG) and behavioral measurements to access retinal function in animals in ways similar to those used to evaluate human vision in the clinic. These studies address human conditions of retinal and macular degenerations and age-related macular degeneration. Using Antibodies To Investigate Retinal Pathophysiology: We found that inherited photoreceptor degeneration in the Royal College of Surgeons (RCS) rat is accompanied by an aberrant increase in the electroretinogram (ERG) response originating in neurons at least two synapses away from degenerating photoreceptors. ERG responses in these cells depend on potassium channel activity in associated glial cells. We developed a novel method for blocking specific membrane channels, including glial potassium channels, and receptors in the retina using antibodies. Using potassium channel antibodies injected in vivo, we explored the contribution of these channels to the aberrant ERG responses of the RCS rat and implicated potassium channel activity in their generation. Simultaneously, we could localize these antibodies to particular retinal cell types and subcellular locations. This approach provides a powerful opportunity to probe mechanisms of disease states using antibodies and non-invasive ERG response recordings to probe pathophysiologic mechanisms in vivo, even during treatment. Rod And Cone Pathway Interactions In Mutant Mice Lacking Rod Function: Retinal visual function depends on rod and cone photoreceptors and associated neural pathways. We are studying the cone pathway responses in mutant mice that lack functional rods due to knockout of either of two genes: NRL, which is necessary for the development of rods, or rhodopsin, which is necessary for the development of their photoreceptive membrane and capacity to respond to light. Both have been shown to still have anatomically intact rod pathways. We have found time course and amplitude changes in the cone pathway responses during light exposure in these mutant mice that, in normal mice, have been attributed to light activation of the rod pathway. Our data indicates that rods without photoreceptive membranes constitutively activate the rod pathway, and that when rods are replaced by cones (in the NRL knockout mouse) the cones activate the normal rod pathway. These results show that (1) the rod pathway that develops in the absence of functional rods is physiologically intact, which could be important for a therapy that replaces the non functioning rods and (2) cones that develop in place of rods in the NRL knockout model signal through the rod pathway, which is a physiological demonstration of retinal plasticity, the ability to modify neuronal connections in response to developmental and disease changes. Gene Therapy with Retinal Survival Factors: In addition to therapy for retinal degenerations by replacement a specific mutated gene , this year we completed a study oof viral vector mediated over expression of endogenous retinal growth and survival factors lens epithelial derived growth factor (LEDGF) and heat shock protein 27 (HSP27) in slowing retinal degeneration in rat models. These factors could be useful as therapy for a broad range of retinal degenerations. We cloned each of these genes into viral vectors for retinal delivery into the intact rodent eye. Both agents slowed the natural course of degeneration in the RCS rat model of inherited retinal degeneration due to a mutation in the MERTK gene expressed in retinal pigment epithelial cells. We also analyzed the concomitant changes in gene and protein expression in relation to the disease state and treatment with these agents. Photoreceptor Plasticity and Homeostasis In Normal and Diseased Retina: A critical facet of retinal neurodegenerative disease involves the structural changes, particularly to the photoreceptor outer segments (OS) that precede photoreceptor death, causing loss of vision. As photoreceptor cells undergo primary degeneration through progressive outer segment (OS) shortening in many of these conditions, a critical question is whether the outer segment may exhibit sufficient structural plasticity to support elongation of OS that have been shortened by disease states and whether this would promote survival of the photoreceptor cell. The goal of the work is to investigate the molecules that are important in the regulation of OS length under light stress and genetic degenerative conditions. We are focusing on neurotrophic factors, such as CNTF, and on small molecules that regulate cytoskeletal growth, including RAC1. The Role of RAC1 In Oxidative Stress-Induced Retinal Damage: In addition to its role as a cytoskeletal element, RAC1 is a component of NADPH oxidase, which is known to cause oxidative damage in some tissues such as the heart. Some retinal degenerations caused by inherited and environmental factors, such as light, are thought to involve stress-induced oxidative damage. This year we completed a study of light-induced photoreceptor damage in Rac1 conditional knockout mice. As RAC1 knockout is embryonic lethal, we developed a conditional knockout of photoreceptor RAC1 using the Cre-loxP system coupled to the opsin promoter and then crossed these mice with a light damage sensitive strain. Mice with an estimated 50% knockdown in photoreceptor Rac1 had reduced photoreceptor death due to bright light exposure. The knockdown did not affect any other retinal function, including visual acuity, electroretinographic response and visual pigment photochemistry, or development and maintenance of retinal structure. In addition, we analyzed the levels of RAC1 and other components of NADPH oxidase and of light damage pathways during light exposure and found no difference between control and knockout animals except in membrane associated RAC1. This evidence points to Rac1 as a key component of the pathway leading to light-induced photoreceptor death through activation of NADPH oxidase. Since normal retinal function was not affect by the knockdown of RAC1, inhibition of RAC1 may be a viable therapeutic strategy for some types of retinal degeneration.